Session 5: Biochemistry of cancer Oral presentations O5.1

Oxidative stress and cancer Grzegorz Bartosz* Uniwersity of Łódź, Łódź, Poland *e-mail: Grzegorz Bartosz   Numerous clues link carcinogenesis, tumor biology and metastasis with oxidative stress and antioxidant defense. The two-faced action of reactive oxygen (as well as nitrogen and chlorine) species involves both the induction of DNA damage and intermediacy in intracellular signaling cascades. Both these pathways can favor the induction of cancer. Reactive oxygen species can be involved in initiation and promotion of carinogenesis, activation of proto-oncogenes and inactivation of stability and tumorsuppressing genes. They may oxidatively activate chemical carcinogens. Numerous data document induction of carcinogenesis by chronic active inflammation e. g. due to infection by Helicobacter pylori or Schistosoma haematobium. Oxidative stress has been implicated in the mechanism of cancer induction by ethanol. However, there are also cases in which the level of oxidative damage of DNA is increased but it does not lead to increased carcinogenesis. Reactive oxygen species enhance the p53 protein activity but increased p53 activity may enhance ROS production. On the other hand, very high levels of ROS inhibit p53. ROS are involved in the action of several proteins involved in the cell cycle regulation. ROS promote apoptosis but their effects are complex and include inactivation of caspases and activation of the Akt pathway promoting cell survival. ROS may promote metastasis, i. a. by modulating integrin expression and promoting angiogenesis. At least some malignant tumors generate more ROS than normal cells . Cancers have been reported to have low MnSOD activity and mice heterozygous with respect to MnSOD have increased risk of developing many types of tumors. The same refers to deficiencies in other antioxidant enzymes. However, there are also reports showing that high MnSOD is linked to enhanced tumor invasion, metastasis and poor prognosis. Chemotherapy has often been associated with oxidative stress. Enhanced antioxidant mechanisms in tumor cells in vivo have been implicated in chemoresistance and leads to poor prognosis, whereas most in vitro studies have reported tumor-suppressing properties of antioxidant enzymes. Dietary or pharmaceutical antioxidant supplementation has been considered a plausible way to prevent ROS-mediated carcinogenicity. However, many antioxidants can act both as antioxidants and prooxidants. Perhaps due to this fact, antioxidant supplementation, though suggested to decrease cancer incidence, lead to equivocal outcome.

In summary, the interaction of oxidative stress and carcinogenesis is an interesting research field still expected to bring results of considerable importance in taming the cancer development.

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O5.2

O5.3

Aneuploidy and cancer; Bcr-Abl expression leads to compromised mitotic spindle checkpoint promoting chromosomal instability

From chemoresistance to cancer stem cells: the case of BRCP/ABCG2 half transporter

Piwocka1*,

Wolanin1,

Katarzyna Kamila Adriana Magalska1, Susanne Vejda2, Sharon L. McKenna2, Ewa Sikora1 1Nencki

Institute if Experimental Biology PAS, Warszawa, Poland, 2University College Cork, Cork Cancer Research Centre, Cork, Ireland *e-mail: Katarzyna Piwocka   Aneuploidy and chromosomal instability are features of cancers that are usually associated with poor prognosis. Whether aneuploidy is a cause or consequence of cancer and its participation in progression of the disease remains an opened question and is subject of hot scientific debate. Some studies suggest that dysfunction in cell cycle checkpoints leads to genomic instability and contribute to tumor progression. The Bcr-Abl oncoprotein plays a major role in the development of chronic myeloid leukemia (CML). Mechanisms participating in the progression from chronic to blast crisis phase remain largely unknown, however chromosomal instability and aneuploidy are common and are likely to be contributory factors. The mitotic checkpoint is a cell cycle control mechanism leading to the accurate segregation of chromosomes during mitosis. As a compromised spindle checkpoint plays a key role in genetic instability, we investigated the influence of Bcr-Abl on the integrity of the mitotic checkpoint. We showed that Bcr-Abl expression was associated with compromised spindle and tetraploid checkpoints leading to aberrant mitoses, centrosomal abnormalities and spindle defects. Bcr-Abl expressing cells escaped from metaphase arrest upon nocodazole treatment and underwent mitotic slippage associated with p53 and p21 induction and 4NG1 arrest. Cells with a high level of Bcr-Abl expression underwent endoreduplication indicating a compromised tetraploid checkpoint. In addition, we found downregulation of mitotic checkpoint genes (Mad2, Bub1, Bub3 and Aurora B), as well as BRCA1 protein, one of key players involved in the division regulation. We have shown for the first time a direct molecular link between the process of aberrant mitosis followed by aneuploidy and chromosomal instability and the level of Bcr-Abl expression. These data have important implications for chemotherapy, suggesting poor responsiveness to agents targeting spindle checkpoints and further demonstrating the promotion of genomic instability by Bcr-Abl.

Acknowledgements: This work was supported by FEBS Collaborative Experimental Scholarship for Central & Eastern Europe and a grant 2P04A 05729 from the Ministry of Science and Higher Education in Poland.

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Andrzej M. Składanowski* Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, Gdańsk, Poland *e-mail: Andrzej M. Składanowski   Cancer cell resistance to chemotherapy is often associated with overexpression of plasma membrane ABC (ATPbinding cassette) pumps which transport a variety of compounds through the membrane against a concentration gradient by the ATP-dependent mechanism. Most of the ABC transporters, including first discovered P-glycoprotein (ABCB1) consist of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. The ABC transporters constitute a large family of membrane proteins and have substrates of extremely divergent chemical structures, which include natural substances such as lipids, bile acids, peptides for antigen presentation and also exogenous and endogenous toxins (xenobiotics). By promoting active extrusion of these substrates outside cells, they reduce the body load of potentially harmful substances. However, they also eliminate various useful drugs from the body, causing multidrug resistance. It now appears that from many different ABC transporters characterized so far, only P-glycoprotein (ABCB1), MRP1 (ABCC1), MRP2 (ABCC2), and BCRP (ABCG2) are directly associated with the phenomenon of multidrug resistance observed in drug-resistant cell lines. The human ABCG2 is a half-transporter and is postulated to form homodimers in the plasma membrane, which actively extrude a wide variety of chemically unrelated compounds from cells. Physiologically this protein plays a crucial role in protecting cells and tissues from toxic effects of xenobiotics, in particular in the intestine, liver and placenta, and is functionally involved in the blood-brain barrier. Overexpression of ABCG2 protein in many different tumor cell types has been shown to confer multidrug resistance to a variety of anticancer agents, including mitoxantrone, daunorubicin, doxorubicin, topotecan, methotrexate, flavopiridol and epirubicin. However, it has been established that the ABCG2 gene is characteristic for its very commonly occurring single nucleotide polymorphisms, which is associated with changed drug affinity and protein distribution, and single aminoacid substitutions in ABCG2 protein at position 482 greatly influence the substrate specificity of this transporter toward different xenobiotics. More recent studies show that ABCG2 transporter plays a key role in the regulation and protection of stem cells, including a special type of cancer cells which also have the ability to self-renew and are called cancer initiating cells or cancer stem cells. One of the molecular markers associated with cancer initiating cell phenotype is increased expression of ABCG2 protein which could be one of the

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reasons of the inherent resistance of cancer initiating or cancer stem cells to antitumor treatment. The different functions of ABCG2 protein in drug resistance and the regulation of cancer stem cells will be discussed.

2007

O5.4 Tumor blood vessels — a target for anticancer therapy Stanisław Szala* Department of Molecular Biology, Comprehensive Cancer Centre Maria Skłodowska-Curie Memorial Institute, Branch Gliwice, Gliwice, Poland *e-mail: Stanisław Szala   Ever increasing number of data suggests that tumor blood vessels are a convenient target of anticancer therapies. Destroying these vessels leads to the ensuing necrosis of cancer cells found in the vicinity of such damaged vessels. This effect has been intensely exploited in anticancer therapy. Some drugs that are capable of selectively recognizing cells of tumor blood vessels may also feature another distinct functional domain, one that induces apoptosis in these cells. We designed and carried out the synthesis of such a drug. Its tumor blood vessel cell-recognizing domain is formed by VEGF121, whereas the effector, apoptosis-inducing domain is secured by A chain of abrin, a potent plant toxin. Thus, the drug recognizes endothelial cells lining tumor blood vessels, is internalized by them and induces their apoptosis. Therapeutic experiments in rodents demonstrated the drug’s potent tumor-growth inhibitory effect. We believe that this drug can be used in various combined therapeutic strategies also involving conventional chemotherapeutic agents known to specifically destroy cancer cells.

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O5.5 Influence of DNAzymes to β1 integrin subunit on tumoral neovascularisation Izabela Sacewicz*, Magdalena Wiktorska, Tomasz Wysocki, Czesław S. Cierniewski, Jolanta Niewiarowska Department of Molecular and Medical Biophysics, Medical University of Łódź, Łódź, Poland *e-mail: Izabela Sacewicz   Angiogenesis, the development of new blood vessels by the sprouting of the existing vasculature or by endothelium-dependent intussusceptive microvascular growth, occurs in a limited range of healthy adult tissues such as the ovary and endometrium during the menstrual cycle and in wound healing but also occurs in various diseases, including cancer. During tumor growth its cells undergo the transformation to an angiogenic phenotype and malignant cells are capable of inducing proliferation and migration in endothelial cells. Because rapid tumor growth requires its own blood supply, when new vasculature is not adequate tumor become necrotic or apoptotic. Angiogenesis is a complex process that involves extracellular matrix remodeling, endothelial cells migration and proliferation. Cell surface integrins, which are the major receptors for extracellular matrix, participate in all of these processes. They are heterodimeric transmembrane glycoproteins, composed of noncovalently-associated α and β subunits. Until now 24 of integrin receptors are known. They are composed from various combinations of 18 α and 8 β chains. The most abundant subfamily of integrins is that consist of β1 chain. Based on given above reasons DNAzymes to β1 mRNA were synthesized — active and inactive ones. The DNAzyme is single-stranded anti-sense oligodeoxynucleotide with enzymatic activity. The model of DNAzyme has a catalytic domain of 15 highly conserved deoxyribonucleotides, flanked by 2 substrate-recognition domains. To increase the stability of DNAzyme in cells and to protect it from nucleases, modified oligonucleotides with 2’-O-methyl-substituted residues were introduced at two final nucleotides both on the 5’ and 3’ sides. Presented work is showing that gene-inactivating agents — metylated derivative of DNAzymes, can be used to downregulate neovascularization process in mouse models. Angiogenesis was examined using a bFGF supplemented Matrigel plugs in BALB/c mice. The DNAzymes were locally administrated every second day causing reduction in revascularization in Matrigel plugs, which was detected by measurement of haemoglobin level. In Human Tumor Xenograft Models, where BALB/cA nude (nu–/–) — B6.Cg — Foxn1nu athymic mice were used, to subcutaneously created human colorectal and prostatic carcinomas DNAzymes were injected. We provide evidence that the active form of mouse DNAzyme to β1 integrin subunit decreased tumors volume and kinetic of their growth, intensity of neovascularisation process evaluated by measurement of haemoglobin concentration as well as PECAM-1 (Platelet Endothelial Cell Adhe-

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sion Molecul-1, CD31) immunohistochemical staining. In conclusion, these DNAzymes might ultimately provide a therapeutic means to inhibit pathological angiogenesis in tumors.

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O5.6 Epigenetic therapy — modulation of the effects of nucleoside analogues on methylation and expression of selected tumour suppressor genes by natural compounds in breast cancer cells Barbara Krawczyk*, Krystyna Fabianowska-Majewska Department of Biomedical Chemistry, Medical University of Łódź, Łódź, Poland *e-mail: Barbara Krawczyk   Promoter methylation of tumour suppressor genes is involved in silencing of their transcription and can be a target for epigenetic therapy. The aim of the present studies was to examine the effects of combined action of natural compounds [i.e. all-trans retinoic acid (ATRA), vitamin D3 (Vit.D3), resveratrol (RES)] and anticancer agents [i.e. nucleoside analogues: 2-chloro-2’-deoxyadenosine (2CdA), 9-beta-d-arabinofuranosyl-2-fluoroadenine (F-ara-A), 5aza-2’-deoxycytidine (5-aza-dCyd)] on promoter methylation and expression of tumour suppressor genes (i.e. BRCA1, RARbeta2, PTEN, APC) as well as on expression of DNA methyltransferase (DNMT1) and p21 genes in non-invasive MCF-7 and invasive MDA-MB-231 breast cancer cells. Methylation of promoters of the tested genes and expression of the genes on the mRNA level were estimated using methylation-sensitive restriction analysis (MSRA) and real-time PCR, respectively. In MCF-7 cells all tested compounds (at IC50 concentrations) reduced RARbeta2, PTEN and APC promoter methylation, whereas the effects of the tested compounds on the gene expression were more differentiated. PTEN mRNA level increased by 30% in the cases of all compounds, expression of RARbeta2 increased over 2-fold after treatment with nucleoside analogues and APC expression increased by 20–50% after treatment with nucleoside analogues and Vit.D3. In invasive MDA-MB-231 cells, only 5-aza-dCyd (at IC50 concentration) reduced PTEN methylation (by 25%). The reduction of PTEN methylation by other tested compounds required several-fold higher concentrations than IC50. Moreover, the changes of PTEN promoter methylation did not result in alteration of PTEN expression, with the exception of Vit.D3 which presence led to increase of PTEN expression by 35% and slight decrease of DNMT1 expression. Combination of ATRA, Vit.D3 and RES with nucleoside analogues gave varied results among that the most striking were: (i) in MCF-7 cells Vit.D3 significantly enhanced 2CdA effect on PTEN methylation (decrease by 63%) and expression (1.5-fold increase), which was accompanied by 2-fold decrease of DNMT1 and 1.7-fold increase of p21 expressions; (ii) in both cell lines ATRA improved stimulation of RARbeta2 expression by 2CdA and 5-aza-dCyd; (iii) in MCF-7 cells RES enhanced inhibitory effect of Fara-A on RARbeta2 and APC methylation as well as on DNMT1 expression (2-fold); simultaneously we observed an increase of p21 and APC mRNA levels (4.5- and 2-fold, respectively).

2007

Our findings provide evidence that ATRA, Vit.D3 and RES may play an important role in prevention of carcinogenesis. Moreover, the results indicate that natural compounds (Vit.D3 and RES) considerably improved anticancer action of nucleoside analogues only at early stage of cancer progression (i.e. in non-invasive MCF-7 cells) and the fact should be taken into account in epigenetic therapy.

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O5.7

O5.8

Reduction of hCGβ gene expression in HeLa cell line by modified U1 snRNA

Role of HO-1 in chemically induced carcinogenesis in mice

Anna Jankowska1*, Beata B. Burczyńska1, Mirosław Andrusiewicz1, Anna Szczerba1, Samuel Gunderson2, Jerzy B. Warchoł1

Halina M. Waś1*, Aleksandra Sierpniowska1, Małgorzata Sokołowska1, Klaudia Skrzypek1, Paweł Dominik1, Anna Ratajska2, Antoni Prątnicki2, Bożena Lackowska3, Anna GrochotPrzęczek1, Józef Dulak1, Alicja Józkowicz1

1Department

of Cell biology, Poznan University of Medical Sciences Poznań, Poland, 2Department of Molecular Biology, Piscataway, United States *e-mail: Anna Jankowska   Human chorionic gonadotropin (hCG) a sialoglycoprotein hormone composed of two noncovalently linked subunits α (hCGα) and β (hCGβ) is physiologically produced by trophoblastic cells of the placenta. Recent studies demonstrated that besides of placenta the hCG and especially its β-subunit is secreted by malignant trophoblastic tumors, as well as by a varieties of tumors of different origin. The role of hCG in tumorogenesis is unknown but it is suggested that hCGβ can stimulate growth of cancer cells or inhibit the apoptosis. Selective downregulation the expression of particular gene allows researchers to determinate its function in many cellular processes. Targeting the mRNA transcripts of a specific gene with antisense RNA, ribozyme or siRNA has shown variable success. In our recent study we applied an alternative approach for reducing the mRNA output of target gene based on a modified U1 snRNA. Previously we showed that the active hCGβ gene is present in nontrophoblastic gynecological cancers tissue. Here we used the property of U1 snRNA to block the accumulation of specific RNA transcript when it binds to its donor sequence within the terminal exon as an effective method for inactivating beta subunit of human chorionic gonaotropin gene in HeLa cell line. The first 10 nucleotides of the human U1 snRNA gene, which normally binds to 5’ss in pre-mRNA were replaced by a sequence complementary to a 10-nt segment in the terminal exon of the hCGβ mRNA. The 5’ end-muted U1 snRNAs block the expression of hCGβ in cervix carcinoma cell line what was showed by immunohistochemistry. hCG was undetectable in cancer cells expressing U1 constructs. Further analysis showed that knocking down the gonadotropin expression significantly increases the apoptosis rate. Almost 95% all cells expressing a modified U1 sn RNAs showed morphological changes characteristic for apoptotic process. The result of the study showed that the possibility to effectively block the hCGβ expression, could lead to a new way to treat patients with gynecological cancers.

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1Faculty

of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland, 2Department of Pathological Anatomy, Medical University, Warszawa, Poland, 3Department of Pathology, Oncology Center, Kraków, Poland *e-mail: Halina M. Waś   Background: Heme oxygenase-1 (HO-1) is an enzyme catalyzing the reaction of heme degradation to biliverdin, carbon monoxide and iron ions. HO-1 plays an important role in cytoprotection, angiogenesis and regulation of inflammatory response. It has been shown to facilitate the progression of many types of tumors. In our previous experiments we demonstrated that overexpression of HO-1 in tumor cells led to decrease in survival time of melanoma-bearing mice, inhibition of inflammatory reaction, and increase in tumor vascularization and number of metastases in lungs. However, role of HO-1 in tumor initiation has not been investigated yet. Therefore, the aim of this study was to elucidate the role of HO-1 in the first step of tumorigenesis, namely in tumor induction. Methods and results: Induction of cutaneous squamous cell carcinoma (SCC) in mice, by topical chemical [7,12dimethylbenzanthracene (DMBA) and phorbol 12-myristate 13-acetate (PMA)] application, is a multistep process involving papilloma formation and progression to carcinoma. To investigate the role of HO-1 in the tumor initiation this two-step model of chemical carcinogenesis was applied to HO-1 knockout (HO-1–/–) and HO-1 wild type (HO-1+/+) mice. Initiator in this process was 0.1  µM DMBA dissolved in 100 µl of acetone that was applied once to the dorsal shaved skin of each animal. Two weeks later, twice-weekly applications of PMA -5 nmol dissolved in 100 µL of acetone-to the same area began and were continued for 28 weeks. Tumor development was monitored in all mice (10–12 per group). HO-1–/– mice were more sensitive to DMBA/PMA treatment than their wild type littermates (40% versus 73% of alive animals at 28th week). Moreover, tumor initiation was delayed in HO-1+/+ animals in comparison to HO-1–/– animals (from 4th week to 6th week) and all HO-1–/– mice developed tumors till 17th week comparing to 100% of tumor-bearing animals at 26th week in case of HO-1+/+ counterparts. Additionally, HO-1 knockout mice grew bigger tumors than their wild type counterparts and this tendency maintained for whole experiment. Interestingly, there were not differences in number of tumors growing on mice of different genotypes. Furthermore, preliminary data suggest some variation in proinflammatory and proangiogenic cytokines in sera samples and in tumor lysates from HO-1–/– and HO-1+/+ mice as measured by

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ELISA test. G-CSF and VEGFR-1 seemed to be upregulated in sera, whereas VEGF and KC appeared to be increased in tumor lysates from HO-1–/– mice comparing to those from HO-1+/+ mice. On the other hand, TNF and IL-1 showed slight upregulation in tumor lysates from HO-1+/+ mice. Conclusions: HO-1 appears to protect mice against SCC induced by DMBA/PMA treatment as HO-1–/– animals developed tumors earlier than their wild type counterparts. It suggests that despite its permissive proangiogenic and prometastatic activities in the growing tumors, HO-1 can play a protective role in tumor initiation.

2007

Posters P5.1

Polymorphism of DNA repair genes as a cancer risk factor; comparison between head and neck squamous cell carcinoma and colon cancer Karol Jelonek1,2*, Agnieszka GdowiczKłosok1, Monika A. Pietrowska1, Maciej Wideł1, Marcin Zeman1, Mariusz Kryj1, Maria Wideł1, Joanna Rzeszowska-Wolny1,2, Zbigniew Grzywna2, Piotr Widłak1 1Maria

Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland, 2Silesian University of Technology, Gliwice, Poland *e-mail: Karol Jelonek   Proteins involved in mechanisms of DNA repair play a crucial role in modulation of carcinogenic processes. Genes encoding these proteins, so called DNA repair genes, are present in human populations in different polymorphic variants. It is widely accepted that polymorphic variants of these genes are associated with differences in the repair of carcinogen-induced DNA damage and may influence an individual risk of cancer. Here we aimed to assess usefulness of analyses of genetic polymorphism of several DNA repair genes for determination of individual risk for head and neck cancer and colon cancer. We have analyzed two groups of tumor-bearing individuals (50 patients with head and neck squamous cell carcinoma and 50 patients with colon cancer) and healthy controls (100 healthy donors). Genetic polymorphism were determined in genomic DNA isolated from blood cells using PCR-RFLP method. We have analyzed following polymorphic variants: (1) Asp to Asn substitution in codon 312 and (2) Lys to Gln substitution in codon 751 of the protein XPD, (3) Asp to Glu substitution in codon 148 of the APE1 protein, (4) Glu to Gln substitution in codon 185 of the NBS1 protein, (5) Arg to Gln substitution in codon 399 of XRCC1 protein, and (6) Ser to Cys substitution in codon 326 of the hOGG1 protein. To validate significance of detected differences in polymorphic variant distribution between healthy donors and patient groups we used the Fisher-FreemanHalton permutation test. When frequencies of particular genotype variants were compared among different groups several statistically significant differences have been found. We have observed that heterozygotic genotype at XPD 312 (Asp/Asn) and heterozygotic genotype at APE1 148 (Asp/Glu) were more frequent in group of patients with colon cancer as compared to healthy individuals (p = 0.002 and p = 0.003, respectively). The data indicate that these genotypes might be a risk factor for colon cancer (OR = 0.79 and OR = 1.87, respectively). We have also observed that homozygotic genotype at APE1 148 (Glu/Glu) and homozygotic genotype at XRCC1 399 (Gln/Gln) were absent in group of patients with head and neck cancer. This suggest that the presence of the 148 Asp allele of APE1 and the 399 Arg allele of XRCC1

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are risk factors for head and neck cancer (OR = 2.15 and OR = 3.21, respectively). Acknowledgements: The project was supported by the Ministry of Science and Higher Education, grant 2P05B12630.

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P5.2 The role of WWOX in normal and pathological cell metabolism Urszula Lewandowska1*, Elżbieta Płuciennik2, Małgorzata Czyż3, Elżbieta Hrabec1, Andrzej Bednarek2 1Department

of Medical Enzymology, 2Department of Molecular Cancerogenesis, 3Department of Molecular Biology of Cancer, Medical University of Łódź, Łódź, Poland *e-mail: Urszula Lewandowska   Background: Breast carcinigenesis results from changes in gene expression, primary activation of oncogenes and down-regulation of tumour suppressor genes. One of recently discovered genes involved in breast carcinogenesis is WW domain-containing oxidoreductase (WWOX) located on chromosome 16q23.3-24.1. Aim: The aim of the present study was to elucidate the role of tumour suppressor gene WWOX in normal and pathological cell metabolism. Materials and methods: The research was carried out on normal immortalized human mammary epithelial cell line HBL-100 and malignant human mammary epithelial cell line MDA-MB-231. Both cell lines were transduced with WWOX cDNA (WWOXw-t, WWOXΔ6-8 and WWOXantisense) cloned into pLNCX2 retroviral vector. Ectopic WWOX expression was confirmed by means of real-time RT-PCR. The transduced cells were further subjected to the following analyses: soft agar growth assay, Matrigel invasion assay, cell growth in Matrigel, and cell cycle analysis by cytometry. Results and conclusions: The results of our research demonstrated that an increase in WWOX expression in both cell lines dramatically decreased their anchorage-dependent growth in soft agar. For the colonies with diameter from 100 to 400 µm the average number of colonies formed by MDA-MB-231/vector was 127 ± 4, for MDA-MB-231/WWOXΔ6-8 it was 65 ± 21, whereas for MDA-MB-231/WWOXw-t it was only 9 ± 2. In the case of normal cells (HBL-100) the average number of colonies with diameter 100-400 µm was 1669 ± 43 for HBL-100/vector, while for HBL-100/WWOXw-t it was only 660 ± 77. On the contrary to the results obtained in the soft agar assay, Matrigel invasion assay showed that an increase in WWOX expression resulted in higher cell migration through basement membrane. Both MDA-MB231 and HBl-100 cell lines transduced with WWOXw-t cDNA exhibited branching morphogenesis, unlike the cells transduced with empty vector only, which formed spheroids. On the basis of those results, one might presume that WWOX is probably involved in formation of mammary gland structures. Flow cytometric analysis of DNA content revealed that no significant changes in cell cycle were observed in both cell lines transduced either with vector or with WWOX cDNA. Increased aggressiveness had no influence on cell cycle.

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P5.3

P5.4

Decreased level of TSG101 expression in epithelial cells at different stages of cervical cancer development is presumably not regulated by epigenetic modifications

Expression of the YEATS protein family in human cancer tissues

Justyna K. Broniarczyk1*, Michał Łuczak2, Agata Józefiak2, Anna Kwaśniewska3, Witold Kędzia2, Anna Goździcka-Józefiak1 1Adam

Mickiewicz University, Poznań, Poland, 2University of Medical Sciences, Poznań, Poland, 3Medical University of Lublin, Lubin, Poland *e-mail: Justyna K. Broniarczyk   Cervical cancer is one of the most common cancers that affects women in Poland. Human Papillomaviruses (mainly HPV16, 18 and 33)-mediated transformation of epithelial cells has been recognized as a multi-step process, in which, apart from the presence of the virus, additional factors are needed. It has been suggested that TSG101 plays a very important role in tumor formation and progression. TSG101 gene was mapped to chromosome 11 p15.1-p15.2. This region of chromosome is known to be associated with a loss of heterozygosity (LOH) in several tumor types. Although somatic mutations or deletions within the TSG101 gene are rare events, aberrant splicing forms have been observed frequently in a variety of human cancers. TSG101 encodes a multidomain protein involved in a range of biological functions: ubiquitination, transcriptional regulation, endosomal trafficking, proliferation and cell survival. Real-Time PCR analysis showed decreased level of TSG101 mRNA in epithelial cells during different stages of cervical cancer development. Reductions of TSG101 mRNA level might be caused by several different factors such as structural changes (point mutations, deletions) in coding or promoter sequences, by promoter hypermethylation or by changes in the profile of transcription factors involved in TSG101 gene expression. Application of PCR/ SSCP methods demonstrated, that there is no mutation in TSG101 coding sequence. Preliminary studies of CpG island methylation patterns in TSG101 promoter sequence in neoplasia and cancer cells showed minor alterations in comparision to healthy tissue. These results suggest that decreased level of TSG101 mRNA in epithelial cells during different stages of cervical cancer development is neither regulated by epigenetic modifications nor by mutations in coding sequence.

2007

Katarzyna A. Szołtysek*, Jakub Hanus, Michał Jarząb, Piotr Widłak Comprehensive Cancer Centre Maria Skłodowska-Curie Memorial Institute, Branch Gliwice, Gliwice, Poland *e-mail: Katarzyna A. Szołtysek   Yaf9 protein is a S. cerevisiae protein involved in remodeling of chromatin structure but also in controlling stability of the yeast genome. Human ortholog of Yaf9 is called GAS41 and contain a structural domain called YEATS. This domain has been identified in several other human proteins, which comprised YEATS protein family: MLLT1/ENL, MLLT3/AF9 and BRDT. YEATS protein family members are involved in regulation of gene expression, yet their exact functions are not clear at the moment. However, their similarity to yeast Yaf9 suggested involvement in controlling genomic stability and possible importance for cancer development. Here we aimed to address this possibility and analyzed the levels of YEATS protein family transcripts in human cancer tissues and corresponding non-malignant tissues. Data regarding levels of YEATS protein family transcripts have been extracted from global gene expression profiles obtained by mean of expression microarrays. We have examined datasets from Affymetrix microarrays analyzed in Comprehensive Cancer Centre Maria Skłodowska-Curie Memorial Institute, Branch Gliwice or present in publicly available databases. Analyses were performed on material from breast cancer, papillary thyroid cancer, bladder cancer, lung cancer, mesothelioma and melanoma, matched with corresponding non-malignant tissues. We have observed that expression of MLLT3/AF9 gene was two-fold lower in thyroid cancer as compared to non-cancerous thyroid. The change has a high statistical significance (p